Anti ago2 antibody

Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (Millipore) was applied to perform Ago2-RIP assay based on the protocol. Cells were reaped at a confluence of 80–90% and lysed in RIP lysis buffer with magnetic bead conjugated and anti-Ago2 antibody (Millipore) or IgG overnight at 4 °C. After digesting with proteinase K (Absin, Shanghai, China), the immunoprecipitated RNA was purified and analyzed by qRT-PCR.

RIP experiments were performed with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA). The magnetic beads were conjugated with an anti-Ago2 antibody (Millipore) or anti-IgG antibody (Millipore) and then incubated with cell lysates at 4 °C for 8 h. The protein was digested using proteinase K, and purified RNAs were used for the qRT-PCR analysis of circ_0092367 and miR-1206 expression.

RNA immunoprecipitation (RIP) assays were performed as previously described.13 Wild‐type NNT‐AS1 or mutant NNT‐AS1 were synthesized and then cloned into SGC‐7901 cells. SGC‐7901 cells were also co‐transfected with miR‐NC or miR‐424 mimic. Cells were lysed by lysis buffer containing a protease inhibitor cocktail and RNase inhibitor. Magnetic beads were pre‐incubated with an anti‐GFP antibody (Abcam) or anti‐rabbit IgG (Millipore) for 1 hour at room temperature, and lysates were immunoprecipitated with beads at 4°C overnight. After 48 hours, cells were used to perform RIP assay using an anti‐AGO2 antibody (Millipore) as described above.

Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Shanghai, China) was adopted to conduct RIP experiments. The cells were collected and resuspended in RIP lysis buffer (Beyotime, Shanghai, China), and the cell extract was incubated overnight with RIP buffer containing magnetic beads coupled to anti-Ago2 antibody (Millipore, Shanghai, China) or IgG. Subsequently, after washing 3 times, the magnetic beads were incubated with proteinase K at room temperature. Total RNA in the immunoprecipitation was then extracted using TRIzol reagent. Ultimately, the relative abundance of circ_0001287 and miR-21 was measured by qRT-PCR.

miR-145-5p and miR-1-3p mimics and negative control (NC) were purchased from GenePharma (Shanghai, China) and transfected into CHL-1 cells using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s manual. Forty-eight hours after transfection, RIP assays were performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) and anti-AGO2 antibody (Cat# 03-110, 5 μL, Millipore) following the manufacturer’s manual. The enriched RNA was detected by real-time PCR as described above.